title: Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation.
abstract: Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 µM) or a construct expressing antisense U7snRNA (0.25-2 µg) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands;
authors.forename: Francesca
authors.initials: F
authors.lastname: Nuzzo
authors.forename: Claudia
authors.initials: C
authors.lastname: Radu
authors.forename: Marco
authors.initials: M
authors.lastname: Baralle
authors.forename: Luca
authors.initials: L
authors.lastname: Spiezia
authors.forename: Tilman M
authors.initials: TM
authors.lastname: Hackeng
authors.forename: Paolo
authors.initials: P
authors.lastname: Simioni
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Castoldi
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005165:Factor V / Q000235:genetics*; D005166:Factor V Deficiency / Q000097:blood / Q000235:genetics* / Q000628:therapy*; D015316:Genetic Therapy; D056945:Hep G2 Cells; D006720:Homozygote; D006801:Humans; D007438:Introns; D008533:Megakaryocytes / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D012326:RNA Splicing / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics / Q000627:therapeutic use*; D012342:RNA, Small Nuclear / Q000235:genetics / Q000627:therapeutic use; D014162:Transfection
chemical_list: D016372:RNA, Antisense; D012342:RNA, Small Nuclear; C405487:U7 small nuclear RNA; D005165:Factor V